Locating ligand binding and activation of a single antiporter

Abstract

Single-molecule force spectroscopy was applied to unfold individual Na þ /H þ antiporters NhaA from membrane patches. The force-extension curves contained detailed information about the strength and location of molecular interactions established within NhaA. Although molecular interactions that stabilize secondary structure elements remained unaffected on switching NhaA into its functional state, those that are assigned to the Na þ -binding site changed markedly. These interactions were formed only in the presence of Na þ , with their full strength being established at pHE6. This finding is in apparent contrast to measurements that suggest that NhaA is fully active at pH 7. Statistical analysis, however, showed that not all NhaA molecules activated this molecular interaction at pH 6, but at pH 7. This implies that the molecular interactions established on Na þ binding may represent an early step in NhaA activation. The direct observation of molecular interactions established within an antiporter provides new insights into their activation mechanisms.

Characterizing folding, structure, molecular interactions and ligand gated activation of single sodium/proton antiporters

“…These molecular interactions are collectively formed by all aa in a segment and can be related to local structural features, such as single helical domains (Müller et al 2002c), helical kinks ), Pi-interactions (Cisneros et al 2005). It was, however, also possible to detect molecular interactions originating from protein-protein interactions (Sapra et al 2005) and ligand binding and protein activation (Kedrov et al 2005a). …”

“…To reveal insights into these mechanisms, SMFS was applied to characterize molecular interactions of inactive and active NhaA forms (Kedrov et al 2005a). Firstly, the force spectra showed that the stability of structure segments established within single antiporters was unaffected upon the activation.…”

“…Recent measurements on the G-protein coupled receptor (GPCR) rhodopsin allowed the assigning of the structural and functional role of such localized molecular interactions stabilizing and holding highly conserved residues at their location (Sapra et al, submitted). That these molecular interactions have not just a mechanistic rule stabilizing the protein and its substructures, were shown by recent experiments in which the binding of a ligand activating the antiporter NhaA could be detected and located as well (Kedrov et al 2005a). (Müller et al 2002a).…”

“…Once the force externally applied by the SMFS reaches a certain critical value, all aa within these segments unfold cooperatively. Initial experiments allowed investigation into how environmental variations such as temperature changes (Janovjak et al 2003), pH variations (Müller et al 2002c;Kedrov et al 2005a), or oligomeric assembly (Sapra et al 2005;Janovjak et al 2005a) influence the strength of molecular interactions establishing stable structural segments within the protein. It has become possible to directly observe the various pathways and to determine the folding steps and kinetics of a polypeptide folding into the final antiporter.…”

“…Comparing the molecular interactions established within two different membrane proteins having almost identical structures allowed insights into their origin (Cisneros et al 2005). Comparative experiments detecting molecular interactions of a membrane protein being in different well-defined functional states reveals insights into molecular mechanisms associated with its activation (Kedrov et al 2005a). Recent measurements on the G-protein coupled receptor (GPCR) rhodopsin allowed the assigning of the structural and functional role of such localized molecular interactions stabilizing and holding highly conserved residues at their location (Sapra et al, submitted).…”